Screening methods involving the detection of short-lived proteins

ABSTRACT

A method is provided for screening for agents that affect protein degradation rates, the method comprising: taking a library of cells, the cells expressing a fusion protein comprising a reporter protein and a protein encoded by a sequence from a cDNA library derived from a sample of cells, the sequence from the cDNA library varying within the cell library; contacting the library of cells with a plurality of agents which may affect protein degradation rates; for each agent, selecting cells in the library which express short-lived proteins based on whether the cells have different reporter signal intensities than other cells in the library, the difference being indicative of the selected cells expressing shorter lived fusion proteins than the fusion proteins expressed by the other cells in the library; and characterizing the fusion proteins expressed by the selected cells for each agent.

CROSS-REFERENCE

This application is a continuation application of Ser. No. 12/804,733,filed Jul. 27, 2010, which is a continuation application of Ser. No.11/303,037, filed Dec. 14, 2005 (now U.S. Pat. No. 7,790,378), which isa continuation application of Ser. No. 10/053,516, filed Jan. 16, 2002(now U.S. Pat. No. 7,056,665). This application claims priority to andbenefit of a each of these applications. Each of these applications isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention The present invention relates to detecting andcharacterizing proteins and more specifically to detecting andcharacterizing short-lived proteins.

2. Description of Related Art

The availability of the entire human genome sequence will revolutionizethe way biology and medicine will be explored in the next century andbeyond. However, the next big challenge is the development oftechnologies for the comprehensive analysis of gene expression and theinterpretation of the functionality of individual genes and their geneproducts in the human genome.

A gene is genetic information (i.e., DNA or RNA) that encodes a protein.Proteins, the expression product of genes, have different biologicalfunctions within a cell. For example, proteins may act as enzymes,interact with DNA or protein, contribute to the cellular skeleton orpossess some other function.

Unfortunately, it is difficult to predict the function of most geneproducts directly from their gene sequences. As a result,characterization of the biological function of any individual geneproduct, its association with disease and its pharmaceuticalapplications are all problems that need to be addressed even after agene is identified.

One post-genomics field, proteomics, is attempting to bridge theknowledge gap between gene sequences and their biological functions.However, the difficulties facing proteomics are multifaceted. Unlikegenes that comprise only four nucleotides and a relatively simple doublehelical structure, proteins are polymers that comprise differentcombinations of twenty different amino acids. The amino acid sequence ofa protein affects the structure of the protein and hence its function.Some proteins also undergo post-translational modifications that affecttheir structure and biological activity.

The way in which a protein is expressed also affects the role that theprotein plays within a cell. A protein may be expressed or not expressedin response to different conditions, in response to the presence ofdifferent agents, and at different levels. Where a protein is expressedwithin a cell and where the protein is transported after expression alsoimpact the protein's function.

The degradation rate of a protein both affects and evidences its rolewithin a cell. For example, short-lived proteins, i.e., proteins with ashort half life, are believed to be very important proteins in cells. Ithas been commented that the most important proteins will be shown to beshort-lived and that most short-lived proteins will be shown to beimportant.

Examples of proteins that have already been shown to be short-livedinclude tumor suppressor p53, oncoprotein myc, cyclins, signalingprotein IκB, and key biosynthetic enzymes such as ornithinedecarboxylase. Their rapid turnover makes it possible for their cellularlevel to change promptly when synthesis is increased or reduced.Schimke, R. T. (1973) Control of enzyme levels in mammalian tissues.Advanced Enzymology, 37, 135-187.

It is believed that many proteins that turn over rapidly within cellshave regulatory roles. For example, transcription factors, cell cycleregulators and metabolic enzymes are all believed to be relativelyshort-lived proteins.

Identifying whether a given protein is short-lived is very useful towardidentifying the protein's role within the cell. Unfortunately however,analysis of whether a given protein is short-lived is currentlytime-consuming and labor-intensive. The most definitive form of analysisrequires pulse-chase labeling cells and immunoprecipitating extracts. Invitro assay of degradation is simpler than in vivo analysis, but an invitro assay system is difficult to establish and may not fully mimic thedegradation of proteins in cells.

Identifying which proteins among all the proteins expressed by a cellare short-lived is highly desirable since it may serve to identify whichproteins are the more important proteins to study. However, genome-widefunctional screening and systemic. Characterization of cellularshort-lived proteins is more complicated than analyzing the lifetime ofa single known protein. Identification of short-lived proteins is moredifficult because they are degraded more rapidly and tend to be presentin lower quantities within the cell. Short-lived proteins are thusharder to detect, isolate and characterize. A need currently exists fora technology that allows for high throughput screening of whetherproteins are short-lived.

SUMMARY OF THE INVENTION

The present invention relates to methods, compositions and kits fordetecting and characterizing short-lived proteins. Through the presentinvention, it is possible to perform genome-wide functional screeningand systemic characterization of cellular short-lived proteins.

According to one embodiment, a method is provided for selecting cellsbased on whether the cells express a short-lived-protein, the methodcomprising: taking a library of cells, the cells in the libraryexpressing a fusion protein comprising a reporter protein and a proteinencoded by a sequence from a cDNA library derived from a sample ofcells, the sequence from the cDNA library varying within the celllibrary; modifying a rate of protein expression or degradation by cellsin the library; and selecting a population of cells from the library ofcells based on the population of cells having different reporter signalintensities than other cells in the library, the difference beingindicative of the population of cells expressing shorter lived fusionproteins than the fusion proteins expressed by the other cells in thelibrary.

According to another embodiment, a method is provided for selectingcells based on whether the cells express a short-lived protein, themethod comprising: taking a library of cells, the cells in the libraryexpressing a first reporter protein and a fusion protein comprising asecond reporter protein and a protein encoded by a sequence from a cDNAlibrary derived from a sample of cells, the sequence from the cDNAlibrary varying within the cell library; modifying a rate of proteinexpression or degradation by cells in the library; and selecting apopulation of the cells from the library of cells based on whether thecells have a different normalized reporter signal intensity than othercells in the library, the normalized reporter signal intensitycomprising a reporter signal from the fusion protein normalized relativeto a reporter signal from the first reporter protein, the differencebeing indicative of the population of cells expressing shorter livedfusion proteins than the fusion proteins expressed by the other cells inthe library.

According to yet another embodiment, a method is provided for selectingcells based on whether the cells express a short-lived protein, themethod comprising: taking a library of cells, the cells in the libraryexpressing a fusion protein comprising a reporter protein and a proteinencoded by a sequence from a cDNA library derived from a sample ofcells, the sequence from the cDNA library varying within the celllibrary; partitioning the library of cells into populations of cellsbased on an intensity of a reporter signal from the fusion protein suchthat cells partitioned into a given population have a reporter signalwithin a range of reporter signal intensity; modifying a rate of proteinexpression or degradation by cells for a given population of cells; andselecting a subpopulation of cells from the given population of cellsbased on whether the cells have a different reporter signal intensitythan the other cells in the given population, the difference beingindicative of the subpopulation of cells expressing shorter lived fusionproteins than the fusion proteins expressed by the other cells in thegiven population.

According to yet another embodiment, a method is provided for selectingcells based on whether the cells express a short-lived protein, themethod comprising: taking a library of cells, the cells in the libraryexpressing a first reporter protein and a fusion protein comprising asecond reporter protein and a protein encoded by a sequence from a cDNAlibrary derived from a sample of cells, the sequence from the cDNAlibrary varying within the cell library; partitioning the library ofcells into populations of cells based on an intensity of a reportersignal from the fusion protein such that cells partitioned into a givenpopulation have a reporter signal within a range of reporter signalintensity; modifying a rate of protein expression or degradation bycells for a given population of cells; and selecting a subpopulation ofthe cells from the population of cells based on whether the cells have adifferent normalized reporter signal intensity than the other cells inthe population, the normalized reporter signal intensity comprising areporter signal from the fusion protein normalized relative to areporter signal from the first reporter protein, the difference beingindicative of the subpopulation of cells expressing shorter lived fusionproteins than the fusion proteins expressed by the other cells in thegiven population.

According to another embodiment, a method is provided for selectingcells based on whether the cells express a short-lived protein, themethod comprising: forming a construct library encoding a library offusion proteins, the fusion proteins comprising a reporter protein and aprotein encoded by a sequence from a cDNA library derived from a sampleof cells; transducing or transfecting the construct library into cellsto form a library of cells which express the library of the fusionproteins; screening the transduced or transfected cells for cells whichexpress the fusion protein; partitioning the screened cells intopopulations of cells based on an intensity of a reporter signal from thefusion protein such that cells partitioned into a given population havea reporter signal within a range of reporter signal intensity; modifyinga rate of protein expression or degradation by cells in the givenpopulation; and selecting a subpopulation of the cells from the givenpopulation of cells based on whether the cells have a different reportersignal intensity than the other cells in the given population, thedifference being indicative of the subpopulation of cells expressingshorter lived fusion proteins than the fusion proteins expressed by theother cells in the given population.

According to this method, the library of cells may optionally furtherexpress an internal standard protein having a different reporter signalthan the reporter protein, and selecting the subpopulation of cells mayoptionally further comprise normalizing the reporter signal from thefusion protein using the reporter signal from the internal standardprotein.

According any of the above methods, screening may be performed using aflow cytometer. In such instances, the reporter protein is preferably aprotein that can be detected by the flow cytometer and used to screenthe cells.

According any of the above methods, the reporter protein may be afluorescent protein. For example, the reporter protein may be a greenfluorescence protein (GFP), an enhanced green fluorescence protein(EGFP), or a red fluorescent protein. The reporter protein may also bebeta-galactosidase.

According any of the above methods, screening and partitioning may beperformed using a flow cytometer.

Also according any of the above methods, when the reporter protein is afluorescent protein and partitioning is performed, the range of reportersignal intensity is optionally a half log interval of fluorescence.

Also according any of the above methods, when the reporter protein is afluorescent protein and partitioning is performed, a given populationthat is formed may optionally have a modal brightness that differs fromanother population by a factor of at least 3.

Also according any of the above methods, when the reporter protein is afluorescent protein and partitioning is performed, partitioning maycomprise partitioning the screened cells into at least 4 populations ofcells where the reporter signal intensities of cells within a givenpopulation do not overlap with the reporter signal intensities of cellswithin another population of cells.

Also according any of the above methods, when protein expression isinhibited, selecting a subpopulation of the cells from the givenpopulation of cells may be based on cells having a reduced reportersignal intensity than the other cells in the given population.

Also according any of the above methods, when protein expression isinhibited, selecting a subpopulation of the cells from the givenpopulation of cells may be based on cells having less than half reportersignal intensity than the other cells in the given population.

Also according any of the above methods, when protein degradation isinhibited, selecting a subpopulation of the cells from the givenpopulation of cells may be based on cells having an increased reportersignal intensity than the other cells in the given population.

Also according any of the above methods, when protein degradation isinhibited, selecting a subpopulation of the cells from the givenpopulation of cells may be based on cells having more than twice thereporter signal intensity than the other cells in the given population.

Also according any of the above methods, the selected subpopulation ofthe cells may optionally be subjected to one or more additional roundsof selection, each round of selection comprising modifying a rate ofprotein expression or degradation by the cells, and selecting a furthersubpopulation of the cells based on whether the cells having a differentreporter signal intensity than the other cells in the given population.

Also according any of the above methods, the selected subpopulation ofthe cells may optionally be subjected to one or more additional roundsof selection such that at least one round of selection comprisesinhibiting protein expression and at least one round of selectioncomprises inhibiting protein degradation.

Also according any of the above methods, the selected subpopulation ofcells may optionally be further selected, at least partially, byculturing cells separately and individually monitoring how the reportersignal of each cell changes in response to protein synthesis or proteindegradation being inhibited.

Also according any of the above methods, the selected subpopulation ofcells may optionally be further selected, at least partially, byculturing cells separately and individually monitoring how the reportersignal of each cell changes using a fluorescent plate reader.

Also according any of the above methods, the methods may optionallyfurther comprise analyzing whether the fusion protein of the selectedcells is short-lived by a pulse-chase analysis.

Also according any of the above methods, the method may optionallyfurther comprise analyzing whether the fusion protein of the selectedcells is short-lived by radiolabelling the expressed fusion protein;immunoprecipitating the expressed fusion protein with anti-GFP antisera;and analyzing the immunoprecipitate by SDS-PAGE and autoradiography.

Also according any of the above methods, the method may optionallyfurther comprise determining the nucleic acid sequences of the fusionproteins.

Also according any of the above methods, the method may optionallyfurther comprise determining the protein sequences of the fusionproteins.

Also according any of the above methods, the method may optionallyfurther comprise analyzing whether the portion of the fusion proteinencoded by the sequence from the cDNA library is short-lived whenexpressed independent of the reporter protein.

Methods are also provided for monitoring the effects that differentgrowth conditions have on expression of short-lived proteins

In one embodiment, the method comprises: exposing samples of cells todifferent growth conditions; forming cDNA, libraries from the sample ofcells after exposure to the different growth conditions; forming alibrary of cells for each cDNA library, the cells in the libraryexpressing a fusion protein comprising a reporter protein and a proteinencoded by a sequence from the cDNA library derived from a sample ofcells, the sequence from the cDNA library varying within the celllibrary; for each library of cells: identifying cells within the librarythat express fusion proteins that are degraded in vivo more rapidly thanother fusion proteins, and characterizing fusion proteins expressed bythe identified cells; and comparing which fusion proteins arecharacterized for each library of cells, differences in thecharacterized fusion proteins indicating differences in the short-livedproteins expressed by when the cells are exposed to the differentagents.

In one variation, identifying cells within the library that expressfusion proteins that are degraded in vivo more rapidly than other fusionproteins comprises modifying a rate of protein expression or degradationby the cells, and selecting a population of the cells based on whetherthe cells have a different reporter signal intensity than the othercells after the rate of protein expression or degradation has beenmodified.

In another embodiment, the method comprises: exposing samples of cellsto different conditions; forming cDNA libraries from the sample of cellsafter exposure to the different growth conditions; forming a library ofcells for each cDNA library, the cells in the library expressing afusion protein comprising a reporter protein and a protein encoded by asequence from the cDNA library derived from a sample of cells, thesequence from the cDNA library varying within the cell library; for eachlibrary of cells: partitioning the library of cells into populations ofcells based on an intensity of a reporter signal from the fusion proteinsuch that cells partitioned into a given population have a reportersignal within a range of reporter signal intensity, modifying a rate ofprotein expression or degradation by the cells for a given population ofcells, selecting a subpopulation of the cells from the given populationof cells based on whether the cells have a different reporter signalintensity than the other cells in the given population, andcharacterizing fusion proteins expressed by at least a portion of theselected cells; and comparing which fusion proteins are characterizedfor each library of cells, differences in the characterized fusionproteins indicating differences in the short-lived proteins expressed bywhen the cells are exposed to the different agents.

In one variation, exposing the samples of cells to different conditionscomprises exposing the cells to different agents.

A method is also provided for screening for differences in short-livedproteins expressed by first and second cell samples.

In one embodiment, the method comprises: forming cDNA libraries forfirst and second samples of cells; forming a library of cells for eachcDNA library, the cells in the library expressing a fusion proteincomprising a reporter protein and a protein encoded by a sequence fromthe cDNA library derived from a sample of cells, the sequence from thecDNA library varying within the cell library; for each library of cells:identifying cells within the library that express fusion proteins thatare degraded in vivo more rapidly than other fusion proteins, andcharacterizing fusion proteins expressed by the identified cells; andcomparing which fusion proteins are characterized for each library ofcells, differences in the characterized fusion proteins indicatingdifferences in the short-lived proteins expressed by the first andsecond samples cells.

In another embodiment, the method comprises: forming cDNA libraries forfirst and second samples of cells; forming a library of tells for eachcDNA library, the cells in the library expressing a fusion proteincomprising a reporter protein and a protein encoded by a sequence fromthe cDNA library derived from a sample of cells, the sequence from thecDNA library varying within the cell library; for each library of cells:partitioning the library of cells into populations of cells based on anintensity of a reporter signal from the fusion protein such that cellspartitioned into a given population have a reporter signal within arange of reporter signal intensity, modifying a rate of proteinexpression or degradation by the cells for a given population of cells,selecting a subpopulation of the cells based on whether the cells have adifferent reporter signal intensity than other cells after the rate ofprotein expression or degradation has been modified, and characterizingfusion proteins expressed by at least a portion of the selected cells;and comparing which fusion proteins are characterized for each libraryof cells, differences in the characterized fusion proteins indicatingdifferences in the short-lived proteins expressed by the first andsecond samples cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a general overview of how short-lived proteins encodedby DNA from a cDNA library may be detected and characterized in ahigh-throughput manner according to the present invention.

FIG. 2A illustrates a process of inhibiting either protein expression ordegradation and then screening for a subpopulation of cells that have adifferent reporter protein signal.

FIG. 2B illustrates exemplary fluorescence intensity plots for theprocess illustrated in FIG. 2A.

FIG. 3 illustrates a method for monitoring how degradation rates ofdifferent proteins change under different conditions.

FIG. 4 illustrates an embodiment of a method for comparing whichshort-lived proteins are expressed by two or more different samples ofcells.

DETAILED DESCRIPTION OF THE INVENTION

Proteins that degrade more rapidly than other proteins in vivo (i.e.,proteins with short half lives) are believed to be functionallysignificant and hence proteins whose study should be prioritized. Byidentifying these proteins and better understanding their function andhow their expression and degradation are regulated, a myriad oftherapeutic applications can be developed. For example, it may provetherapeutically advantageous to induce or inhibit expression of certainof these proteins for selected disease states. It may also provetherapeutically advantageous to develop inhibitors for certain of theseproteins for selected disease states. It may also prove therapeuticallyadvantageous for certain disease states to increase or decrease the halflife of these proteins in vivo, for example by stimulating or inhibitingthe regulatory pathway controlling the degradation of these proteins.

As will be described herein, the present invention provides highthroughput methods that allow short-lived proteins to be identified andstudied more efficiently. For example, the present invention relates tomethods for identifying which proteins expressed by a given cell sampleare degraded more rapidly than other proteins also expressed by the cellsample. The more rapidly degraded proteins are referred to herein as“short-lived proteins.” By understanding which proteins are short-lived,these proteins may be targeted for further study. Expression of at leastsome short-lived proteins is regulated. The present invention alsorelates to methods for identifying short-lived proteins whose expressionis affected by particular conditions. By knowing what conditions affectthe expression of different short-lived proteins, therapeuticapplications may be developed to induce or inhibit their expression.

The degradation rate of some proteins may also be regulated. The presentinvention relates to methods for identifying short-lived proteins whosedegradation rate in vivo is affected by particular conditions. Byknowing what conditions affect the degradation of different short-livedproteins, how protein degradation of particular short-lived proteins isregulated can be better understood. Further, therapeutic applicationscan be developed as a result of better understanding bow degradation ofthese proteins is regulated and what agents influence their degradation.

Compositions and kits for use in combination with the various methods ofthe present invention are also provided.

Advantageously, the methods of the present invention are high-throughputmethods in the sense that they can be used to perform genome-widefunctional screening and systemic characterization of groups of cellularproteins as short-lived proteins. Because short-lived proteins arelikely to be functionally significant, the ability to systematicallyidentify certain proteins as being short-lived greatly assists inidentifying which are the more important proteins being expressed. Giventhat many short-lived proteins are regulatory proteins, knowing whichproteins are short-lived also helps to determine the functionalsignificance of these proteins.

Using the technology of the present invention, functional identificationof important regulatory proteins from the entire human genome is madepossible in a high-throughput screening format. With this technology,human genes can be systematically screened and new genes can easily beidentified from expression libraries. Because of their importance inbiological function, these short-lived proteins have a great potentialin drug discovery.

As will become evident by the following description of the invention,the methods of the invention advantageously allow one to differentiateand identify short-lived proteins from longer lived proteins withoutknowing in advance which proteins are short-lived and without knowing inadvance the sequences of the various short-lived proteins that willultimately be identified.

FIG. 1 provides a general overview of how short-lived proteins may bedetected and characterized in a high-throughput manner according to thepresent invention.

As illustrated, mRNA 101 is obtained from a cell sample 100. A cDNAlibrary 102 is then formed from the mRNA 101. The cDNA library 102 and asequence encoding a reporter protein 104 are combined to form aconstruct library 106 encoding fusion proteins, each fusion proteincomprising a protein encoded by a sequence from the cDNA library and thereporter protein.

A vector library 108 is formed from the construct library 106 in orderto introduce the fusion protein constructs into a cell line.Introduction of the vector library may be performed by transduction ortransfection, depending on the nature of the vector and the nature ofthe cell line.

A library of cells 110, once formed using the vector library, expressthe library of fusion proteins. The library of expressed fusion proteinscomprise short-lived fusion proteins and a larger number of longer-livedfusion proteins. Described herein is a process for selecting cells fromthe library that express fusion proteins that behave as short-livedproteins over the larger group of cells that express fusion proteinsthat behave as longer-lived proteins.

As seen in step 112, the fusion proteins are expressed by the library ofcells. The cells are then screened 114 for expression of the fusionprotein based on detection of the reporter signal. The screen 114 servesto remove cells that do not exhibit a reporter signal. As a result,cells that express a fusion protein are separated from cells that eitherdid not receive a construct or received a non-productive construct.

The reporter protein should be a protein whose expression may bedetected in vivo. A variety of such proteins may be used, most commonlyfluorescent proteins such as green fluorescence protein (GFP) andenhanced green fluorescence protein (EGFP) which may be readily detectedand used to screen the cells by a flow cytometer.

After the cell library is screened 114, the screened cells arepartitioned 115 into populations of cells where the measured reportersignal from the fusion protein in a given population is within apredetermined range. For example, if the reporter is fluorescent, thecells are grouped into populations where all the cells in a givenpopulation fluoresce within a given range of fluorescence intensity.

For a given population of cells, the rate at which protein expression ordegradation occurs is then modified 116. A subpopulation of the cells isthen selected 118 from the given population of cells based on, thosecells having different reporter signal intensities than the other cellsin the given population, the difference in reporter signal intensitiesbeing indicative of the subpopulation of cells expressing shorter livedfusion proteins than the fusion proteins expressed by the other cells inthe given population. The subpopulation of cells selected will typicallyrepresent a minority of the cells of the given population.

The process of partitioning the cells into populations 115, modifyingthe rate of protein expression or degradation 116, and selecting asubpopulation of cells based on reporter signal intensity 118 isdescribed in more detail in regard to FIGS. 2A and 2B.

Referring to partitioning the cells into populations 115, FIG. 2Billustrates a plot of fluorescence for cells expressing fusion proteinswhere the reporter is fluorescent. As illustrated, the different cellshave a range of fluorescence intensities 210. In order to better monitorchanges in fluorescence intensities for individual cells, the cells arefractionated into populations of cells where cells in a given populationare all within a narrower range of fluorescence. For example, thefluorescence plot of one fractionated population of cells 212 is shownin FIG. 2B.

Referring to the step of modifying the rate of protein expression ordegradation 116 of FIG. 1, it is noted that short-lived proteins aredegraded faster than other proteins. As a result, when proteinexpression is inhibited, the concentration of short-lived protein in thecell will decrease at a more rapid rate than longer-lived proteinsbecause protein expression is not replacing the short-lived proteins. Asa result, the reporter signal intensity in cells expressing ashort-lived fusion protein will decrease more rapidly than other cellswithin a given population. Referring to FIG. 2A, it is possible toinhibit protein expression 202 and then select cells 206 expressing ashort-lived fusion protein by selecting those cells whose reportersignal is lower than other cells in the cell population. Exemplaryfluorescence intensity plots for this process are illustrated in FIG. 2Bwhere a population of cells that initially had a common fluorescenceintensity (as shown in plot 212) has separated over time into twopopulations where a small sub-population has a lower fluorescenceintensity after protein synthesis is inhibited (as shown in plot 214).

When protein degradation is inhibited in step 116 of FIG. 1, becauseshort-lived proteins are degraded faster than other proteins, theconcentration of short-lived proteins will increase at a more rapid ratethan will longer-lived proteins. As a result, the reporter signal ofcells expressing a fusion protein comprising a short-lived proteinwithin a given population will increase more rapidly than cellsexpressing a fusion protein comprising a longer-lived protein. Referringagain to FIG. 2A, it is possible to inhibit protein degradation 204 andthen select those cells 208 that express a short-lived fusion protein byselecting those cells whose reporter signal is higher than other cellsin the cell population. Exemplary fluorescence intensity plots for thisprocess are illustrated in FIG. 2B where a population of cells thatinitially had a common fluorescence intensity (as shown in plot 212) hasseparated over time into two populations where a small sub-populationhas a higher fluorescence intensity after protein degradation isinhibited (as shown in plot 216).

As illustrated in FIGS. 1 and 2A, the process of inhibiting eitherprotein expression or degradation and then screening for a subpopulationof cells which have a different reporter protein signal may be performedonce or repeated one or more times in order to more carefully selectcells expressing short-lived fusion proteins. For example, in onevariation, at least one selection is performed after inhibiting proteinexpression and at least one selection is performed after inhibitingprotein degradation.

Optionally, the cells selected as having a different reporter signalthan other cells in the population in response to protein synthesis orprotein degradation being inhibited may be further evaluated prior tosequencing the fusion proteins. For example, as described herein,different cells may be cultured separately and then individuallymonitored for how their reporter signal changes in response to proteinsynthesis or protein degradation being inhibited. By monitoring thereporter signal behavior of different cells separately, it is possibleto more carefully evaluate whether a given fusion protein is beingdegraded as would a protein with a relatively shorter half life. As aresult, a more careful cell selection may be performed.

After cells believed to encode short-lived fusion proteins are finallyselected, the nucleic acid and protein sequences of the fusion proteinsmay be determined.

Once the sequences of the fusion proteins and the cDNA encoding them areknown, a variety of additional analyses may be performed. For example,database searches may be performed based on the cDNA or proteinsequences in order to determine whether the cDNA sequence and/or theprotein encoded by the cDNA sequence are already known. In someinstances, the proteins identified by the above selection process willbe novel. Even if some of the proteins are already known, their cDNAsequences may not have been known. Furthermore, the fact that theseproteins are degraded more rapidly is valuable information since itindicates that these proteins may be regulatory proteins.

As can be seen from the above description, the process of the presentinvention allows one to screen an entire cDNA library for proteins whosedifference in degradation rates evidence that these proteins areshort-lived. The proteins and their cDNA need not be known prior toperforming the process of the present invention or known even whenperforming the process. Rather, only those proteins that are likely tobe short-lived proteins need to be sequenced according to the presentinvention.

As can also be seen, the method of the present invention allows thediscovery of various valuable pieces of information that allincrementally help to fill the proteomics knowledge gap.

By being able to rapidly identify proteins as being short-lived incombination with the cDNA sequences encoding the proteins, a myriad ofapplications arise, some of which are described herein in furtherdetail. For example, by determining which proteins are short-lived,arrays comprising cDNA for the short-lived proteins can be producedwhich allow one to rapidly monitor how expression of differentshort-lived proteins changes under different conditions.

The design, operation and applications for the present invention willnow be described in greater detail.

1. Formation of Reporter-cDNA Fusion Protein Construct Library

In order to systematically clone all genes whose products may beshort-lived, a fusion expression library is formed by combining asequence encoding a reporter protein with a cDNA library formed frommRNAs isolated from a sample of cells. A wide variety of methods areknown in the art for forming a cDNA library from mRNA isolated from acell sample. Any of these methods may be used in the present invention.

In one embodiment, an agent such as Trizol reagent (Gibco BRL) is usedto isolate total RNA from cells or a tissue sample. Oligo (dT) columnsis then used to purify poly (A)⁴ RNAs. First-strand cDNA synthesis maythen be primed from poly (A)⁺RNAs by oligo dT primers. A cDNA librarymay then be constructed using SMART (Switching Mechanism at 5′end of RNAtemplate) library construction technology from CLONTECH. This methodsimultaneously employs the two intrinsic properties of M-MLV, namelyRT—reverse transcription of mRNA template and template switchingactivity. The technique allows two different restriction sites to beadded to the anchor and oligo dT primers, to conduct directional cloningcDNAs.

Optionally, the oligo(dT) primer may include an BamH I site and an EcoRI site may be introduced into the anchor. First strand synthesis is thenperformed with 5-methyl dCTP, producing hemimethylated cDNA, with theunmethylated BamH I site on the linker/primer. Second-strand cDNA isgenerated with the unmethylated EcoR I site on the anchor as a primer,using an enzyme mixture of E. coli DNA polymerase, RNA ligase and RNaseH. The double-stranded cDNA is digested with appropriate restrictionenzymes to generate two different sticky ends. After size fractionation,the cDNA may be directionally cloned into expression vectors. Comparedto cDNA cloned nondirectionally, libraries made according to this methodare more likely to make functional fusion proteins for expressionscreening.

The reporter protein may be any protein that enables cells expressingthe reporter protein as part of a fusion protein to be screened in vivo.The sequence encoding the reporter protein may be 3′ or 5′ relative tothe sequence from the cDNA library.

In one embodiment, the reporter protein is an autofluorescent protein. Aunique feature of autofluorescent proteins is their ability to bedetected without any substrate or cofactor. Using an autofluorescentprotein as the reporter, fluorescence associated with single cells canbe analyzed by fluorescence activated cell sorting (FACS), a technologyeasily adapted to high throughput screening. Galbraith, D. W., Anderson,M. T. and Herzenberg, L. A. (1999) Flow cytometric analysis and FACSsorting of cells based on GFP accumulation. Methods Cell Biol, 58,315-41. Thus, FACS can be used for analysis of the large number of humangenes.

Green fluorescent protein (GFP) is an example of an autofluorescentprotein. GFP from the jellyfish Aequorea victoria has been widely usedto study gene expression and protein localization. Tsien, R. Y. (1998)The green fluorescent protein. Annu Rev Biochem, 67, 509-44. GFP hasalso been found in a variety of other organisms including Renilla.

Enhanced GFP (EGFP) is a mutant of GFP with 35-fold increase influorescence, which dramatically improves the detection of GFP. Thefluorescence of GFP is dependent on the key sequence Ser-Tyr-Gly (aminoacids 65 to 67) that undergoes spontaneous oxidation to form a cyclizedchromophore. Enhanced GFP (EGFP) contains mutations of Ser to Thr atamino acid 65 and Phe to Leu at position 64, and is encoded by a genewith human-optimized codons. Cormack, B. P., Valdivia, R. H. and Falkow,S. (1996) FACS-optimized mutants of the green fluorescent protein (GFP).Gene, 173, 33-8.

A wide variety of methods are known in the art for forming a fusionprotein library between a first protein (in this case the reporterprotein) and sequences from the cDNA library. In one embodiment, thefusion protein libraries are constructed by fusing cDNA to the Cterminus of the reporter protein, such as GFP or EGFP. Optionally,pEGFP-N1, N2, and N3 (CLONTECH) may be used to express GFP fusionproteins. pEGFP-N1, N2, and N3 are a set of vectors with three openreading frames. The vectors contain the CMV promoter, multiple cloningsites (MCS), the EGFP gene and an SV40 poly A site. The MCS with threereading frames allows genes to be cloned 5′ relative to the EGFP gene.The expression vectors also contain the SV40 origin of replication,which allows extra-chromosomal replication and facilitate recovery fromcells, such as COS-7, that express the SV40 large T antigen.

2. Formation of Vector Library Comprising Reporter-cDNA Fusion ProteinConstructs

A variety of different vectors may be formed to transfer the library ofconstructs into a cell line. These vectors may introduce the constructsinto the cell line by transfection or transduction. For example, thelibrary of constructs may be ligated into expression vectors such aspd1EGFP, pd2EGFP, and pd4EGFP which are each commercially availablemammalian expression vectors that code for the fluorescence proteinEGFP. These constructs are made from pEGFP-C1 with the C-terminal fusionof the degradation domain of mouse ornithine decarboxylase anddemonstrated in cells with a short half-life, a range from 1 hour to 4hours. To normalize the transfection, a second reporter construct, suchas beta-galactosidase, can be co-transfected with the fluorescenceprotein construct under the control of the same or a different promoter.

3. Formation of Library of Cells Comprising Reporter-cDNA Fusion ProteinConstructs

The library of vectors encoding the reporter-cDNA fusion proteins arethen introduced into a cell line to produce a library of cells whichexpress the reporter-cDNA fusion proteins. Preferably, the cell libraryformed has a diversity of at least >10⁴, more preferably >10⁵, and mostpreferably a diversity of at least >10⁶.

The recipient cell line of the vector library is preferably of a samegenus as the sample of cells from which the cDNA library is derived. Forexample, a fusion protein library formed from cDNA derived frommammalian cells is preferably formed in a mammalian cell line.Similarly, a fusion protein library comprising cDNA derived from plantcells is preferably formed in a plant cell line.

In one embodiment, when the cDNA library is derived from a mammaliancells, the recipient cell line of the vector library is CHO cells orCOS-7 cells. When a pd2EGFP vector is employed, it is desirable to useCOS-7 cells because these cells express the SV40 large T antigen whichresults in high-copy extra-chromosomal replication of the pd2EGFPvector.

Once the library of cells is formed, the library is allowed to expressthe fusion proteins and is then screened for whether the fusion proteinis being expressed. For example, when the reporter is a fluorescentprotein, such as GFP or EGFP, the cells can be efficiently screened byFACS sorting. This allows one to easily separate transformed ortransfected cells from untransformed or untransfected cells and cellsthat were transformed or transformed by non-productive constructs.

4. Sorting Cell Library Into Populations Based on Reporter SignalIntensity

The library of cells formed by transfecting or transducing a cell linewith vectors encoding a library of fusion proteins will have adistribution of reporter signal intensities. For example, when thereporter is a fluorescent protein, a cell population with anapproximately log-normal fluorescence histogram distribution may have afluorescence distribution of 4 logs to the base 10.

According to the present invention, cells that are likely to encodeshort-lived proteins are selected by detecting changes in the cells'reporter signal intensity over time. By narrowing the distribution ofreporter signal intensities within a given population of cells, it ispossible to detect changes in the reporter signal intensities ofindividual cells within the population of cells. Therefore, prior toinhibiting protein synthesis or protein degradation, the cell library isfirst divided into populations, each with a distinct and narrowdistribution of reporter signal intensities. Together, the populationscover the full dynamic range of the library of cells. In one variation,the cell library is divided into 2, 3, 4, 5, 6, 7, 8, 9, 10 or morepopulations.

When a fluorescent reporter protein is employed, FACS fractionation maybe used to divide the library into separate populations where eachpopulation has a distinct and narrow fluorescence brightnessdistribution. Optionally, each population may be fractionated to withina half-log interval of fluorescence. This would cause each population tohave a modal brightness that differs from that of an immediatelyadjacent population by a factor of about 3.3.

After the library is divided into separate populations with a narrowerdistribution of reporter signal intensities than the library, thedistribution of reporter signal intensities for each population may bechecked to confirm that the cells in a given population have the desireddistribution of reporter signal intensities. If the population is notfound to have the desired reporter signal intensity distribution, thepopulation may be fractioned again. This process may be repeated as manytimes as necessary in order to produce populations of cells which eachhave the desired distribution of reporter signal intensities within thepopulation.

5. Selecting Cells By Inhibiting Protein Expression and/or ProteinDegradation

Once separate populations of cells are formed, each population isseparately analyzed for the presence of short-lived proteins.

For a given population, a subpopulation of cells is selected based ontime-dependent changes in the reporter signal intensity of the cellswithin the population in response to inhibiting either protein synthesisor protein degradation. This selection process may be repeated multipletimes where the subpopulation of cells formed in a given round isfurther screened and narrowed in a later selection round. Optionally,the multiple rounds of selection include inhibiting protein synthesisand protein degradation in separate rounds. When both types ofinhibition are performed in separate selections, a finer screen isaccomplished.

In one embodiment, cells that have been partitioned into a population ofcells having a desired distribution of reporter signal intensities areselected based on how inhibition of protein synthesis reduces thereporter signal intensity. A variety of different agents may be used toinhibit protein synthesis. Examples of such agents include, but are notlimited to cycloheximide.

When protein synthesis is reduced or blocked, short-lived proteins aremore readily degraded. Hence, the signal of the reporter in the fusionprotein decreases. By selecting those cells whose reporter signaldecreases more rapidly than other cells, one is able to detect cellsexpressing a short-lived fusion protein.

In one embodiment, cells that have been partitioned into a population ofcells having a desired distribution of reporter signal intensities areselected based on how inhibition of protein degradation increases thereporter signal intensity. A variety of different protein degradationinhibiters may be used. One such inhibitor is lactacystin, a specificproteasome inhibitor. Fenteany, G., Standaert, R. F., Lane, W. S., Choi,S., Corey, E. J. and Schreiber, S. L. (1995) Inhibition of proteasomeactivities and subunit-specific amino-terminal threoriine modificationby lactacystin. Science, 268, 726-731; Omura, S., Fujimoto, T., Otoguro,K., Matsuzaki, K., Moriguchi, R., Tanaka, H. and Sasaki, Y. (1991)Lactacystin, a novel microbial metabolite, induces neuritogenesis ofneuroblastoma cells. J Antibiot (Tokyo), 44, 113-6.

When degradation of short-lived proteins is inhibited, the concentrationof short-lived proteins increases within the cell. This results in thesignal of the reporter in the fusion protein increasing. By selectingthose cells whose reporter signal increases more rapidly than othercells, one is able to detect cells expressing a fusion proteincomprising a short-lived protein.

Exposure to agents that inhibit protein synthesis and proteindegradation should be controlled so that live cells may be recovered andfurther processed. Hence, exposure to inhibitors should be limited todurations that are consistent with survival. Also, it is recognized thatprolonged exposure could induce a secondary cellular response thatproduces alterations in signal intensity from causes other than proteinturnover. This could result in a false-positive background. As discussedherein, a second reporter protein may be used as an internal standard tocounter these potential alterations in reporter signal intensity.

The duration desirable for inhibiting protein synthesis or proteindegradation is dependent upon how great a change in the signal intensityof the reporter is to be detected. It is also dependent upon the desiredmaximum half life of the proteins to be detected. For example, cells maybe selected which show at least a 2×, 4×, 6×, or 8× change in reportersignal intensity. This change in reporter signal intensity may occurover varying lengths of time, such as within 1 hour, 2 hours, 3 hours,etc. In the case of inhibiting protein synthesis, the half life of aprotein would be expected to equal the time required for the reportersignal intensity associated with the protein to decrease by 50%,assuming no pharmacological lag. Hence, a protein with 2 times lessreporter signal intensity after an hour would be expected to have a halflife of about 1 hour. Similarly, a protein with 4 times less reportersignal intensity after two hours and a protein with 8 times lessreporter signal intensity after three hours would both be expected tohave a half life of about 1 hour, assuming no pharmacological lag.

As described above, prior to inhibiting protein synthesis or proteindegradation, the cell library is divided into populations, each with adistinct and narrow distribution of reporter signal intensities. When afluorescent reporter protein is used, each population will have adistinct and narrow fluorescence brightness distribution. Together, thepopulations cover the full dynamic range of the library of cells.

Each population is subjected individually to one or more proteinsynthesis or protein degradation inhibitor selections. For eachselection, cells are selected from the population which by theirreporter signal intensity behave differently than a main portion of thepopulation. For example, cells may be selected from the population whichfall outside of the mean reporter signal intensity for the population bya factor of two, three, four, five, ten or more.

The subpopulation of cells selected after each round of selection isexpected to constitute a very small fraction of the cell populationprior to the selection.

Cells that are selected during each selection round are washed free ofthe protein synthesis or protein degradation inhibitor and allowed toregenerate through cell division in culture. After regeneration, thecells may be subjected to further rounds of selection.

Gene recovery and sequence analysis may be performed on cells selectedafter one or more rounds of selection in order to identify the fusionprotein expressed by the selected cells. Gene recovery and sequenceanalysis may be performed by any of a large number of well-knowntechniques.

6. Optional Further Selection of Cells

The selection process described in Section 5 serves to enrich thepercentage of cells in the resulting population of selected cells thatencode a short-lived protein. Optionally, further selection may beperformed where individual clones of the selected cells are furtheranalyzed for whether they encode a short-lived protein.

According to this variation, the selected cells are separated such thatsingle cells are seeded into wells of microliter plates and allowed togrow, preferably to at least 10⁴ cells per well. The wells may then betreated with a protein synthesis or protein degradation inhibitor.Afterward, the individual wells are scanned to assess time-dependentchanges in the reporter signal. Wells exhibiting time-dependent changesindicative of the cells expressing short-lived proteins may be markedand the cells contained therein recovered. Gene recovery and sequenceanalysis may then be performed on the recovered cells.

This additional selection of individual clones can be carried outmanually with the aid of a fluorescent plate reader. Higher throughputmay be desirable or even necessary if large numbers of cells need to bescreened, for example, because the selection process yields a smallpopulation of desired cells. High throughput screening may be carriedout using a Cellomics ArrayScan Kinetics HCS Workstation (Cellomics,Pittsburgh).

7. Validation of Selection Process

In order to validate the specificity of the selection process, cellsthat are selected may be analyzed using conventional methods to evaluateprotein lability. For example, pulse-chase analysis may be performed toconfirm whether the fusion protein expressed by the selected cells areshort-lived. When GFP is used as the reporter protein, this validationmay be performed by immunoprecipitating the labeled fusion protein withanti-GFP antisera, followed by SDS-PAGE and autoradiography.

8. Internal Standard For Monitoring Selection Efficiency

Stochastic cellular processes can induce the fluorescence signals ofsome cells to change over time. For example, changes in cell shape, cellcycle position, or intracellular redistribution of a fusion protein canall cause the fluorescent signal of a cell to change. When selectingcells based on a change in fluorescence, false positives may be selectedlithe fluorescence signals of those cells change in a manner that causesthe cells to be mistakenly selected as expressing short-lived fusionproteins.

Multiple rounds of population-based selections using FACS will serve toeliminate false positives misidentified as a result of such randomfluctuations. False positive selections will also be eliminated insubsequent, more individualized screens.

It is nevertheless desirable to reduce the frequency with which falsepositives are at least initially selected. This can be achieved by usingan internal standard whose signal also varies as a result of thesestochastic cellular processes. As a result, by normalizing the reporterrelative to the internal standard, a normalized reporter value can bedetermined that is more reliably indicative of the expression of thereporter.

For example, cells may be transformed or transfected so they express afusion protein comprising the first reporter protein and a secondreporter protein, such as beta-galactosidase, that has a differentemission wavelength than the first reporter protein. This allowsexpression of the first reporter protein and the second reporter proteinto be independently monitored. It also allows the signal from the firstreporter protein for each cell to be normalized relative to the secondreporter protein. The normalized reporter signal for a given cell shouldbe less effected by the stochastic cellular processes of that cell.Hence, basing selection upon the normalized reporter signals for eachcell should reduce the frequency of false positives.

The second reporter protein may be introduced into cells by any mannerand by any vehicle. For example, the second reporter protein may also beintroduced into the cell by transformation or transfection and may beintroduced before, after, or with the introduction of the vectorencoding the fusion protein.

In one embodiment, the vector library comprising the first reporter—cDNAfusion protein constructs further encodes the second reporter protein.Hence, initial selection of cells for whether the cells received avector from the vector library may be based either upon the firstreporter protein or the second reporter protein.

Optionally, cells may be added to each population which express a knownshort-lived protein as a benchmark. These benchmark cells for eachpopulation should have a brightness mode that is close to that of itsrelated population. The benchmark cells may be added in knownconcentrations, for example in numbers that constitute 1:100, 1:1000 or1:10,000 of total cells. The benchmark cells may also be marked with abenchmark reporter protein, such as beta-galactosidase. Since othercells in the population will not express the benchmark reporter protein,the effectiveness of the present invention to enrich the concentrationof short-lived proteins relative to the initial cell library can bemonitored by measuring the frequency of this marker.

9. Characterizing Sequence From cDNA Library in Selected Cells

After selecting cells whose reporter signal behavior indicates that thefusion protein is short-lived, the sequences encoding the fusion proteinmay be analyzed. Specifically, the selected cells may be pooled andextra-chromosomal DNA extracted and transfected into E. coli. It isnoted that other methods may be used to recover the gene inserts. Forexample, the gene inserts can be recovered through PCR, using flankingsequences from the vector used to introduce the sequence encoding thefusion protein as a primer.

The E. coli library produced by transfecting the extra-chromosomal DNAmay then be used to obtain DNA sequence information. Individualbacterial cells may be isolated and cultured in commercially available384-well high-density culture plates. Each individual culture plate maybe bar-coded where individual clones are assigned a particular code.This allows the cell lines to be readily retrieved for further analysis.The barcode system may be implemented throughout the entire process.

E. coli cells in replica plates are diluted and used for DNAamplification in an appropriate 384-well PCR plate. After PCRamplification, the DNA fragments can be used for direct sequencing. ADNA sequence database may be established based on the sequenceinformation. The DNA sequence and putative translated protein sequencecan then be examined and compared with existing DNA sequence databaseusing The National Center for Biotechnology Information (NCBI) and byusing the BLAST program run by NCBI, or by The Protein ExtractionDescription and Analysis Tool (PDANT) program. Genes identified that areof interest may be readily retrieved from the original cell clones basedon their barcodes.

10. Confirmation of Whether Isolated Proteins Are Short-Lived in NativeForm

Once the DNA and protein sequences of the fusion proteins areidentified, further analysis may be performed to evaluate whether theportion of the fusion protein encoded by the sequence from the cDNAlibrary is short-lived in its native form, that is, when expressed freeof the reporter protein. Testing of the lability of the native form ofthe protein screened via the above process may be performed by standardmethods, such as pulse-chase analysis, which are known in the art.

11. Monitoring Changes in Degradation Rate of Proteins Under DifferentConditions

It is noted that the degradation rate of a given protein is itselfsubject to regulation. Hence, different proteins may be short-livedunder certain cellular conditions and less labile under otherconditions. FOr instance, IκB, the inhibitor of NFκB, forms a complexwith NFκB and inhibits NFκB activity. When the pathway is triggered byTNF or IL-1, a cascade of kinases in the NFκB pathway is activated,which results in phosphorylation and degradation of IκB. NFκB isreleased from the complex and translocates from the cytoplasm to nucleusto mediate transcriptional induction of a number of genes whose productsare very important to immunity and inflammatory responses.

A need thus exists for methodology that allows one to monitor howdegradation rates of different proteins change under differentconditions.

FIG. 3 illustrates a method for monitoring how degradation rates ofdifferent proteins change under different conditions. According to thisvariation, a library of cells expressing a fusion protein library isformed 110, screened 114 and partitioned 115 according to the presentinvention.

One or more of the partitioned populations of cells 308 is then grownunder different conditions 310A-310C which may serve to regulate proteindegradation. These different conditions may include cell cycle position,inducing conditions or other factors. For example, the differentconditions may include exposing the cells to a library of agents thatmay affect regulation of the degradation process.

Those cells that are found to have a reporter signal behavior indicativeof a fusion protein being degraded as a short-lived protein are selected312A-312C. The selection process may comprise the one or more selectionrounds and other selection processes described above.

The fusion proteins expressed by the selected populations of cells312A-312C are then compared 314. By seeing, which fusion proteins areexpressed by the same population of cells 308, it is possible todetermine how the different conditions influence protein degradation.

By comparing which proteins are degraded by the cells under differentgrowth conditions and when exposed to different agents, the process ofhow the degradation of certain proteins is regulated can be elucidated.For example, by determining that a given protein is labile within a cellin the presence of a given agent but is otherwise a stable protein, oneis able to begin to deduce how that protein is regulated. Thisinformation could lead to the identification and development oftherapeutic agents that either reduce or increase the half life ofselected proteins by knowing how to control the degradation regulatorypathway associated with that protein.

In some instances, conditions may affect the protein degradation of agroup of proteins. By determining groups of proteins that appear to havetheir degradation rate linked in some way, regulatory pathways can bededuced. For example, the fact that administering an agent affects thedegradation of a group of proteins may indicate that the agent is eitherinhibiting or inducing a given pathway. This allows the proteinsinvolved in that pathway to be identified. By finding agents thatinhibit different subgroups of proteins, the pathway may be furtherelucidated.

Being able to determine whether a given agent affects the degradationrate of more than one protein is very useful in designing therapeutics.For example, the fact that a given agent affects the degradation rate ofmultiple proteins may signal that that agent is not sufficientlyselective and may cause undesirable side affects. The fact that a givenagent affects the degradation rate of multiple proteins may also signalthat that protein is not an attractive target for regulating a givenpathway.

12. Comparing Short-Lived Protein Expression Across Different Samples

In Section 11, it was noted that the degradation rate of a given proteinmay be affected by the conditions under which the cells are grown. Inthat instance, a cDNA library isolated from a single sample is testedunder different conditions.

This section describes how to compare which short-lived proteins areexpressed by different cell samples. When the protein expression ofnormal cells and diseased cells are compared, it may be found thatdifferent short-lived proteins are either expressed or not expressed bythe diseased cells. For example, the diseased cells may comprise agenetic abnormality relative to the normal cells. By comparing whichshort-lived proteins are expressed by normal and diseased cells, it maybe possible to identify one or more short-lived proteins whoseexpression or non-expression account for the diseased cells beingabnormal. Treatments may then be directed to these identifiedshort-lived proteins.

FIG. 4 illustrates an embodiment of a method for comparing whichshort-lived proteins are expressed by two or more different samples ofcells. In FIG. 4, a normal 400A and diseased 400B sample of cells areshown. mRNA libraries 402A, 402B and then cDNA libraries 404A, 404B areformed for the cell samples 400A, 400B. Libraries of constructs 406A,406B, libraries of vectors 408A, 408B, and then libraries of cells 410A,410B are formed based on each cDNA library. The resulting libraries ofcells are then each processed as set forth in FIG. 1 in order toidentify short-lived fusion proteins expressed by each library of cells412A, 412B. By comparing 414 which short-lived fusion proteins areexpressed by each library of cells 410A, 410B, it is possible to detectdifferences between the libraries and hence differences between theshort-lived proteins expressed by the two or more different samples ofcells 400A, 400B.

13. Method for Altering Degradation Rate For Short-Lived Proteins

Proteins differ widely in their lability, ranging from entirely stableto half-lives that measure minutes. In some cases, rapidly degradedproteins have been shown to contain an identifiable “degradationdomain.” Removal of this degradation domain makes such proteins stableand appending this domain to a stable protein changes its stabilitydramatically. Such a degradation domain has been identified in a numberof short-lived proteins, such as the C terminus of mouse ODC. (Li, X.,Stebbins, B., Hoffman, L., Pratt, G., Rechsteiner, M. and Coffino, P.(1996) The N Terminus of Antizyme Promotes Degradation of HeterologousProteins. The Journal of Biological Chemistry, 271, 4441-4446;Loetscher, P., Pratt, G. and Rechsteiner, M. (1991) The C Terminus ofMouse Ornithine Decarboxylase Confers Rapid Degradation on DihydrofolateReductase. The Journal of Biological Chemistry, 266, 11213-11220) andthe destruction box of cyclins (Glotzer, M., Murray, A. W. andKirschner, M. W. (1991) Cyclin is Degraded by the Ubiquitin Pathway.Nature, 349, 132-138).

In some cases, the signal is a primary sequence such as the PESTsequence. Rechsteiner, M. and Rogers, S. W. (1996) PEST Sequences andRegulation by Proteolysis. Trends in Biochemical Sciences, 21, 267-271;Rogers, S., Wells, R. and Rechsteiner, M. (1986) Amino Acid SequencesCommon to Rapidly Degraded Proteins: The PEST Hypothesis. Science, 234,364-368. However, the structural features of such degradation domainsare not sufficiently uniform as to provide a reliable guide toidentifying the general class of labile proteins that interests us here.The major neutral protease responsible for degradation of labileregulatory proteins is the proteasome. Zwickl, P., Voges, D. andBaumeister, W. (1999) The Proteasome: A Macromolecular Assembly Designedfor Controlled Proteolysis. Philos Trans R Soc Lond B Biol Sci, 354,1501-11.

Prior to degradation, most short-lived proteins are covalently coupledto multiple copies of the 76 amino acid protein ubiquitin, a reactioncatalyzed by a series of enzymes. Ciechanover, A. and Schwartz, A. L.(1998) The Ubiquitin-Proteasome Pathway: The Complexity and MyriadFunctions of Proteins Death. Proc Natl Acad Sci USA, 95, 2727-30. Theseubiquitinated proteins are recognized by 26S proteasome and degradedwithin its hollow interior. This system of regulated degradation iscentral to such processes as cell cycle progression, gene transcriptionand processing of antigens. A few proteins have been found to beexceptional. Verma, R. and Deshaies, R. J. (2000) A Proteasome HowdunitThe Case of The Missing Signal. Cell, 101, 341-4. Like ornithinedecarboxylase, they do not require ubiquitin modification fordegradation by the proteasome.

A desirable utility of being able to rapidly and efficiently determinethe sequence of a large number of different short-lived proteins is theprospect of identifying additional degradation domains. By knowing whatdomains affect recognition within the cell that a protein should bedegraded, it is then possible to reengineer proteins either to increaseor decrease their rate of degradation in vivo.

A significant problem in the art relates to the rate at whichtherapeutic proteins administered to the body are cleared. With enhancedknowledge regarding how protein degradation is regulated, for example,by better understanding what are the degradation domains of proteins, itis possible to modify the degradation domains of therapeutic proteins sothat these proteins have longer half lives in the body whenadministered.

14. Compositions and Kits for Use in the Methods of the PresentInvention

A wide variety of compositions and kits may be designed for use incombination with the various methods of the present invention. Variousexamples of these compositions, such as reporter—cDNA fusion proteinconstruct libraries 106, vectors comprising the library of reporter—cDNAfusion protein constructs 108, and library of cells expressing thelibrary of reporter—cDNA fusion proteins 110 have already been describedherein.

It is noted that a variety of kits may be formed which may be used toconstruct these various compositions or which may be used in combinationwith these various compositions for performing aspects of the presentinvention. Several of these kits are described herein. Others will bewell understood by one of ordinary skill in the art.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the compounds, compositions,kits, and methods of the present invention without departing from thespirit or scope of the invention. Thus, it is intended that the presentinvention cover the modifications and variations of this inventionprovided they come within the scope of the appended claims and theirequivalents.

What is claimed is:
 1. A method of identifying proteins degradeddifferently in different cells, the method comprising: providing alibrary of cells by introducing into one or more populations of cellsvectors encoding proteins, wherein the proteins are encoded by sequencesfrom the one or more cDNA libraries; expressing the proteins in the oneor more populations of cells; separating the one or more populations ofcells into a first subgroup of cells and a second subgroup of cells;inhibiting expression of the proteins in the first and second subgroupsof cells; detecting protein degradation in the first and secondsubgroups of cells; and, comparing degradation of one or more proteinsin the first subgroup of cells to the degradation of the one or moreproteins in the second subgroup of cells; thereby identifying proteinswith different degradation profiles in the cells.
 2. The method of claim1, wherein the encoded proteins are fusion proteins comprising areporter.
 3. The method of claim 2, wherein said comparing the proteindegradation comprises identifying a lower reporter signal intensity in asubgroup as compared to that detected in another subgroup of cells,after inhibiting protein expression.
 4. The method of claim 1, whereinsaid detecting comprises detecting a protein degradation rate over aperiod of time.
 5. The method of claim 1, further comprising, theexposing first subgroup of cells and second subgroup of cells todifferent conditions, before said inhibiting of expression.
 6. Themethod of claim 5, wherein the different conditions comprise conditionsselected from the group consisting of: culture conditions, growthconditions, exposure to agents, exposure to a library of agents, andexposure to therapeutic agents.
 7. The method of claim 5, furthercomprising identifying patterns of two or more different proteins thathave their degradation influenced by a condition.
 8. The method of claim1, wherein the one or more cDNA libraries is a cDNA library from asingle type of cells.
 9. The method of claim 1, wherein said comparingcomprises identifying one or more proteins that are short-lived in thefirst subgroup and one or more proteins that are short-lived in thesecond subgroup.
 10. The method of claim 1, wherein the one or more cDNAlibraries comprise two different cDNA libraries.
 11. The method of claim10, wherein the two different cDNA libraries are prepared from differentcells.
 12. The method of claim 11, wherein the different cells comprisehealthy cells and diseased cells.
 13. The method of claim 12, furthercomprising identifying proteins associated with the disease of thediseased cell.
 14. A method of identifying proteins that have theirdegradation linked to a common condition in a cell, the methodcomprising: providing one or more cDNA libraries; providing a library ofcells by introducing into a population of cells vectors encodingproteins, wherein the proteins are encoded by sequences from the one ormore cDNA libraries; expressing the proteins in the population of cells;exposing the population of cells to a culture condition; inhibitingexpression of the proteins in the population of cells; detecting proteindegradation in the population of cells; and, identifying a group ofproteins with degradation influenced by the culture condition; therebyidentifying proteins that have their degradation linked to a commoncondition.
 15. The method of claim 13, wherein the encoded proteins arefusion proteins comprising a reporter.
 16. The method of claim 14,wherein the condition is selected from the group consisting of: growthconditions, exposure to an agent, exposure to a library of agents, andexposure to a therapeutic agent.
 17. The method of claim 14, whereinsaid detecting comprises detecting a protein degradation rate over aperiod of time.
 18. The method of claim 14, wherein said detectingcomprises detecting two or more short-lived proteins.
 19. The method ofclaim 14, further comprising elucidating a regulatory pathway based onthe proteins identified.
 20. A method for identifying differences inshort-lived proteins associated with different cell samples, the methodcomprising: providing a first cDNA library from a sample of first cellsand a second cDNA library from a sample of second cells, which firstcells are different from the second cells; providing a first library ofcells expressing sequences encoded by the first cDNA library; providinga second library of cells expressing sequences encoded by the secondcDNA library; inhibiting expression proteins in the first library ofcells and in the second library of cells; detecting the proteindegradation in the first library of cells and in the second library ofcells; identifying one or more proteins that are short-lived in thefirst library of cells and one or more proteins that are short-lived inthe second library of cells; and comparing the identified short-livedproteins between the first library of cells and second library of cells,thereby identifying short-lived proteins associated with cell sampledifferences.
 21. The method of claim 20, wherein the first cells arehealthy cells and the second cells are diseased cells.
 22. The method ofclaim 20, wherein the encoded proteins are fusion proteins comprising areporter.
 23. The method of claim 20, wherein said detecting comprisesdetecting a protein degradation rate over a period of time.